Irreversible inhibitors of acetylcholinesterase (AChE) are used extensively as insecticides and have also been used as chemical weapons. World-wide stockpiles of these agents for use in chemical warfare are estimated to exceed 200,000 tons (Lejeune et al, 1998). The extreme toxicity of these compounds and their global proliferation has made the development of protective agents and antidotes an important research priority.
AChE inhibitors are derivatives of phosphoric, pyrophosphoric and phosphonic acids. These agents react to form a phosphoester with the serine residue that resides in the active site of the AChE enzyme. This phosphoester form of the enzyme is inactive, although the active enzyme can be regenerated through hydrolysis of the phosphoester.
The toxicity of the organophosphorus agents arises from the stability of the phosphoester intermediate that is formed with the enzyme. As its name implies, AChE functions to cleave the ester linkage of acetylcholine, forming acetic acid and choline. To do this, the enzyme transiently forms an ester with the carboxyl moiety of acetylcholine, releasing choline. The resulting carboxylate ester of the enzyme is inactive. However, this ester undergoes rapid hydrolysis, measured in milliseconds, to regenerate the original, active form of the enzyme. In contrast, the rates of hydrolysis for the phosphate esters of AChE are measured in hours. The reversible inhibitors of AChE that are used clinically generate a carbamylated enzyme and have rates of hydrolysis measured in minutes. Thus, the time required for regeneration of the active enzyme via hydrolysis of the ester linkage of the inactive, acetylated enzyme defines the difference between the enzyme's normal function, reversible inhibition, and irreversible inhibition.
One strategy for developing antidotes to irreversible AChE inhibitors has been the synthesis of highly nucleophilic small molecules capable of efficiently cleaving phosphate esters. Hydroxylamine is one such compound that was demonstrated to significantly increase the rate of hydrolysis of phosphorylated AChE. However, efficient hydrolysis was only achieved at toxic concentrations of hydroxylamine.
To date, this approach has yielded only one compound that has shown clinical efficacy, 2-pyridine aldoxime methylchloride (2-PAM). The oxygen atom of this molecule is part of an oxime functional group. The oxime moiety is a hydroxylamine-like nucleophile formed from the reaction of hydroxylamine with aldehydes or ketones. The effectiveness of this compound is limited by its inability to cross the blood-brain barrier. However, effectiveness is further impaired by an inability to regenerate the enzyme once “aging” has occurred. This latter impediment means that treatment is only effective if administered within a few minutes to a few hours of toxin exposure, depending on the toxin.
Organophosphorus agents, particularly some of the more recent additions to chemical weapons arsenals, have the propensity to become truly irreversible inhibitors through the aging process. This molecular process occurs when, having first reacted with AChE to inactivate the enzyme, a phosphoester bond undergoes cleavage, resulting in an anionic ester which is extremely resistant to hydrolysis. The phosphorylated enzyme which has aged in this way is completely refractory to regeneration by currently available antidotal agents, including 2-PAM. It is this aging process that makes the phosphorus-derived chemical warfare agents, such as sarin, soman, VX and tabun, extremely lethal.
In addition to efforts to produce antidotes, research has focused on protecting against exposure to organophosphorus agents, thereby preventing or moderating their toxic effects. One important strategy has been the use of recombinant enzymes for the biocatalytic degradation of organophosphorus agents. This methodology is being utilized in the development of protective clothing as well as agents for surface or aerial decontamination. In these methodologies, microbial enzymes, such as cholinesterases or organophosphorus hydrolases, are attached to a solid support in a manner that retains some portion of their catalytic activity. Such biocatalytic materials include silicone polymers (Gill 2000) and polyurethane foams (LeJeune 1996, 1999; Cheng 1996).
Peripheral blocking methods, i.e., methods that rely on agents that would intercept nerve agents in the circulatory system before they partition into the central nervous system or muscle, are currently the only choice for protection against organophosphorus agents. The standard technology in use today for protection against nerve agents is based upon the recombinant enzyme, butyrylcholinesterase (BuChE). BuChE rapidly reacts with nerve agents to form an intermediate phosphoester with a serine in the active site much as occurs when these agents react with AChE. Compounding the slow rate of hydrolysis of this intermediate is the inactivation of the enzyme through the process of molecular aging discussed above. Thus, BuChE is essentially an autocatalytic stoichoeometric blocker rather than a true hydrolytic enzyme. Butyrylcholinesterase mutants demonstrate enzymatic turnover, but low turnover, combined with high immunogenicity, an inability to cross the blood-brain barrier and a high equivalent weight ratio (almost 500:1 for butyrylcholinesterase to nerve gas). These factors make it difficult to maintain in vivo enzyme levels sufficient to protect against a lethal dose of a toxin such as a nerve agent.
To date, the above-mentioned shortcomings have not been overcome.